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APPUED MICROBIOLOGY, Aug. 1968, p.

1140-1145 Copyright 1968 American Society for Microbiology

Vol. 16, No. 8 Printed in U.S.A.

Isolation and Characterization of a Celluloseutilizing Bacterium


Y. W. HAN AND V. R. SRINIVASAN Department of Microbiology, Louisiana State University, Baton Rouge, Louisiana 70803

Received for publication 6 May 1968

A cellulose-decomposing aerobic and mesophilic bacterium has been isolated from soils of sugar cane fields. The terminal dilution method was adapted to isolate a single clone of cellulolytic organism from closely related contaminants. The cultural and physiological characteristics of the isolate were studied, and the organism was identified as a member of the genus Cellulomonas. The isolate excreted cellulase into the menstruum, and it hydrolyzed various cellulosic materials producing cellobiose as the final breakdown product in the menstruum. When sugar cane bagasse was properly treated with alkali and heat, the organism could decompose up to 90% of the initial substrate within 5 days. Amino acid analysis of the cell crop revealed a high content of lysine, and the essential amino acid pattern compared favorably with that of Food and Agricultural Organization reference protein.
Cellulose is the most abundant of all naturally

occurring organic compounds, probably comprising at least a third of all the vegetable matter on the earth. The biological degradation of ceUulose has been of paramount importance in the activities of the living systems. Usually, plant products are cycled through animals before man can obtain the necessary nutritive protein from his environment. During this cycling process, the efficiency of conversion of the plant products to animal proteins is dependent upon the digestibility of the cellulosic products by animals. Conversion efficiency can be increased considerably if the forage feed is pretreated with microbial enzymes. Furthermore, because of the explosive increase in world population, it is becoming imperative to investigate possible means of obtaining the essential proteins from sources other than animals. Microbial proteins can be developed as food substitutes suitable for human consumption. An efficient mode of conversion of cellulose to nutritive proteins could involve the selection of appropriate strains of microorganisms capable of growing on treated or biodegraded cellulosic wastes. In this investigation, a cellulolytic bacterium that might be suited to such a process was isolated, and its cultural, physiological, and biochemical characteristics were studied.
MATERIALS AND METHODS Media. Isolation media consisted of mineral salts solutions supplemented with 0.1% yeast extract and a strip of filter paper. The mineral salts solution con-

tained: NaCl, 6.0 g; (NH4)2S04, 1.0 g; KH2PO4, 0.5 K2HPO4, 0.5 g; MgSO4, 0.1 g; CaCl2, 0.1 g; and 1 liter of distilled water. Method of isolation ofcellulolytic organism. Rotting sugar cane stalks and the adjacent soil mixture were obtained from a sugar cane field near the Louisiana State University campus. About 1 g of a mixture of soil and sugar cane debris was inoculated into the isolation medium. After 3 to 7 days of incubation at 30 C on a reciprocal shaker, a patch of a yellow-pigmented material appeared at the liquid-air interface on the filter paper. As soon as the pigmented material appeared, a portion of ifiter paper was transferred with a sterile wire and inoculated into fresh medium. This process was repeated several times to enrich the aerobic and mesophilic cellulose-utilizing organisms. The filter paper from the enriched culture was removed, macerated in a small amount of sterile water, and streaked onto plates containing nutrient agar, carboxymethyl (CM) cellulose-agar, or filter paper-agar (a plate of nutrient agar covered with filter paper). Representatives of the various colonies which developed on each of these media were picked and inoculated into fresh media in test tubes. Tubes showing visual degradation of filter paper were selected and alternatively transferred into liquid and solid media for enrichment and isolation of the cellulolytic organism. Isolated colonies were further purified by the terminal dilution method as follows. A culture which disintegrated the filter paper was allowed to grow to its maximal population, and serial dilutions were made into fresh medium. Approximately 100 tubes of the isolation medium containing filter paper strips were inoculated with a sample from each of the higher dilutions. These tubes were incubated for 7 to 10 days and then examined for evidence of filter paper disintegration. The tubes showing disintegration of filter paper were selected,
g;

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and the dilution and selection procedure was repeated until a clone from a single cell was obtained which was capable of disintegrating the filter paper. The purity of the isolated culture was confirmed by microscopic examination and by colony morphology on several agar plates. Characterization and identification. The isolated culture was subjected to diagnostic tests according to the Manual of Microbiological Methods (9), and Bergey's Manual was used for the identification of the strains. Preparation ofcrude enzyme solution. The organism was grown in a liquid medium containing the basal salts mixture with 0.005% yeast extract. After 4 days of incubation with continuous shaking, cell-free filtrates were prepared by centrifugation at 3,000 X g for 20 min at 4 C. The supernatant phase was collected and used as stock enzyme solution. Preparation of substrates. The following substrates were obtained from various sources: filter paper (Whatman no. 1, W&R Balston Ltd.), cellobiose (Difco), cellulose powder (Whatman chromedia CF11, W&R Balston Ltd.), p-aminobenzoyl (PAB)-cellulose (Bio-Rad Laboratories, Richmond, Calif.), CM cellulose (Carl Schleicher & Schull Co., Keene, N.H.), cotton fiber (Johnson & Johnson, New Brunswick, N.J.), paper towel (Garland, soft-knit, single-folded towel, Fort Howard Paper Co., Green Bay, Wis.). Azo-cellulose was prepared by treating PAB-cellulose with sodium nitrite and coupling the diazotized cellulose with B-naphthol to form a red-colored cellulose. Bagasse pith, bagasse fibers, and a alkali-treated and untreated sorgo bagasse were provided by the Department of Chemical Engineering, Louisiana State University. Visual determination of cellulolytic activity. The organism was inoculated into test tubes containing isolation medium and incubated at 30 C. The visual degradation of filter paper was checked at frequent intervals. The breakage of filter paper usually occurred at the air-liquid interface. The visual determination was used as a preliminary screening test for the cellulolytic organisms. Gravimetric determination of cellulolytic activity. The organism was inoculated into basal media containing a limited amount of various cellulosic substrates. After a predesignated incubation period, cultures were filtered through Gooch crucibles (Pyrex, fritted, 30-ml capacity, M porosity). The residue on the crucible containing undigested cellulose residue was washed according to the procedure of Lembeck et al. (5).A control flask was prepared which contained an equal amount of substrate inoculated with a heatkilled culture. The extent of decomposition of the cellulose was calculated by comparing the weight loss of the test flask to that of the control after both samples had been dried to a constant weight at 105 C. Colorimetric determination of cellulolytic activity. The amount of solubilized substrate after the action of the culture or the enzyme solution was determined by the phenol-sulfruic acid method of Dubois et al. (2). After a predetermined incubation period, the reaction mixture was centrifuged or filtered to remove the residual insoluble substrate. The amount of

solubilized substrate was determined on samples of the supernatant fluid or filtrate. Paper chromatography. The enzyme hydrolysate was passed through an ion-exchange resin column (Dowex 2-X8), and the effluent was taken to dryness by evaporation. The residue was dissolved in an appropriate amount of water. About 10 to 20 ,uliters of sample was streaked on Whatman no. 1 filter paper. The sample was then developed in an ascending manner with a solvent (isopropanol-pyridine-acetic acid-water, 8:8:1:4, v/v). Satisfactory separation was obtained after 24-hr development. For detection of nonreducing sugars, the method of Cifonelli et al. (1) was used. The paper was removed, dried, and sprayed with a saturated solution of potassium meta-periodate. After 6 min, the periodate chromatogram was sprayed with a benzidine solution (0.1 M benzidine in 50% aqueous alcohol-acetone-0.2 N HCl, 10:2:1, v/v). Carbohydrates were located by the appearance of colorless spots on a blue background. Reducing sugars were detected according to Stahl (10) by spraying the chromatogram with an aniline phthalate solution (0.93 g of aniline and 1.6 g of phthalic acid dissolved in 100 ml of n-butyl alcohol saturated with water) and heating for 10 min at 120 C. Reducing sugars were visible as red, maroon, or brown spots in a white background.

RESULTS AND DIscussIoN With conventional enrichment and plating techniques, the isolation of pure cultures of cellulose-utilizing organisms was extremely difficult, because the insolubility of cellulose in aqueous media prevents the formulation of proper solid media for isolation. Therefore, many attempts have been made to replace the cellulose with some other soluble substrate, such as cellobiose and cellulose derivatives. After a series of repeated enrichments and platings on these media containing a cellulose derivative, a relatively pure culture of a cellulose-utilizing organism was obtained. However, microscopic examination revealed that the isolate consisted of at least two or three different species of bacteria which were always associated on the solid media used. Thus, it was extremely difficult to separate them by ordinary enrichment and plating techniques. A clone of a single ceU capable of hydrolyzing cellulose was finally obtained by applying the terminal dilution method described in the Materials and Methods. This method permitted successful separation of even a minority of species from a mixed culture by a combination of dilution and

selective cultivation. The isolated strain of cellulose-utilizing organism was a small gram-negative, nonmotile, rodshaped bacterium. It grew on nutrient agar, but not vigorously, and developed small bluish transparent colonies. It was catalase-positive and was capable of hydrolyzing gelatin slowly and attacking cellulose. These characteristics were identical

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to those in the description of genus Cellulomonas medium would parallel the increase of total proin Bergey's Manual. All of the 10 species in the tein during the stationary phase. Since the acgenus Cellulomonas are motile except C. flavigena, tivity shows a steady level, it is highly probable C. uda, and C. acidula. The last-named species is that the enzyme excretion occurs only during a not a nitrate reducer. Therefore, only the descrip- short period of the early stationary phase. The tions of the first two species fit that of the ioslated decline in activity of a later stage could be acstrain on the basis of the available information. counted for either by the inherent instability of In Table 1, the morphological and cultural the enzyme or by the destruction of the enzyme characteristics of the isolated strain were com- by the extracellular proteolytic enzyme activity. As shown in Fig. 2, the maximal enzyme acpared with the description of the two similar tivity, measured by the amount of solubilized species described in Bergey's Manual. The isolated organism excreted cellulase into cellulose, was observed in the pH region of 4.7 to the menstruum. As can be seen in Fig. 1, the 6.8. The optimal pH range for the growth of the activity of enzyme in the menstruum sharply in- organism was noted in the pH region of 6 to 8. creased during the early stationary phase of This difference could be selectively utilized for growth of the organism and showed a decrease on enzyme assay in which a prolonged incubation further incubation. The level of excreted total period was required. For instance, the enzyme protein in the menstruum, however, increased activity could be measured at pH 5.0, although no continuously during the stationary phase of cell significant growth of the organism occurred at growth. If the cellulase were stable and elaborated that hydrogen ion concentration. The organism could utilize many kinds of cellucontinuously, the activity of the enzyme in the
TABLE 1. Descriptive chart of cellulose-utilizing organisms
Characteristic
C. flavigena C. uda

Isolate

Morphological characteristics
Form Size

Rods, curved

0.4-0.6, X 0.7-1.8,u
Nonmotile Variable

Motility Gram stain Cultural characteristics Agar slant

Rods 0.5t X1.0-1.51ju X Nonmotile Negative

Rods, short 0.3-0.5 A X 0.7-1.2 ju Nonmotile Negative

Smooth, glistening opaque, yellow Broth Uniformly turbid Gelatin stab Slow liquefaction Filter paper in pep- Fibers separate on tone broth slight agitation Optimal tempera- 28-33 C
ture Agar colonies

Moderate, flat, grayish white Uniformly turbid Slow liquefaction Fibers separate on sliglht agitation 28-33 C

Smooth, glistening opaque, yellow Uniformly turbid Slow liquefaction Fibers separate on slight agitation 25-35 C Bluish, transparent, smooth, flat, circular, grow feebly on nutrient
agar

Biochemical characteristics Starch Nitrate Methyl red test Voges-Proskauer


test

Hydrolyzed Reduce to NO2

Hydrolyzed Reduce to NO2 Negative


Acid Acid Acid Acid
Acid

Negative
Acid Acid Acid Acid

Hydrolyzed Reduce to NO2 Negative Negative

Indole production Glucose Lactose Sucrose Maltose Dextrin Starch

Negative Acid Acid Acid


Acid

Acid

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degradation. Table 3 shows the effectiveness of alkali treatment on the digestibility of bagasse 10 by the organism. As the concentration of alkali 0-_Viable Colls and the reaction time increased, the digestibility I :4 1 snlog also increased proportionally. Therefore, some /4 sort of physical or chemical pretreatment of natural cellulose may be necessary for the organ0 I an m Ii Proteicn a wi ~~~~~~~~~xism to utilize it effectively. By means of paper-partition chromatography, w~ ~ ~ ~ ~ ~. 30 ~ ~ ~ ~ ~ ~ ~ ~~3.00 an attempt was made to determine whether glucose or cellobiose was the end product of cellulose p p 0 107 2.0 20O'~ degradation by the exocellular enzymes of the 10 40 foe 50 6C7C090 20 3 Oi Cellulose 0 organism. Only cellobiose and intermediary nonactivity reducing glycosides were found (Fig. 3). Glucose > l0 ~~~~~~ ~~~~1.0 z
0.0

~~

~~

w -.1

5.0

0 10 20 30 40 weeicbtdwt0

50

70 90 100 0 60 go 80itrppri

mlo

TABLE 2. Digestibility of various cellulosic materials by the organism


Residual wt
mg

INCUBATION

TIME

HOURS)

FIG. 1. Growth, excretion of protein, and activity of Initial Substrate wt extracellular cellulase of the organism. The activity of enzyme was expressed in terms of solubilized cellulose which is determined by phenol-sulfuric acid method and mg presented as optical density units. Amounts of 2 ml of Filter paper ................ 90.6 culture filtrate, taken at intervals during the growth were incubated with SO mg offilter paper in 4 ml of Cellulose powder ........... 97.0 PAB cellulose .............. 92.9 phosphate buffer (pH 5.91) for 24 hr at 30 C. CM cellulose ............... 91.8 Azo-cellulose ............... 93.0 Cotton fiber ................ 99.4 Paper towel ................ 65.4 73.0 Bagasse pith........ Bagasse fibers .............. 90.0 Sorgo bagasse .............. 109.2 Alkali-treated sorgo Call population 100.0 bagasse...........
-i

Degree
of digestiona

40.7 64.0 54.2 66.0 74.0 99.4 46.0 65.4 87.0 93.0

55.0 34.0 41.0 28.0 20.5 0 30.0 10.0 3.4 15.0

20.0 80.0

I0 4

03
b-

OD

140 120

0.1

z 0
4.

S41

100
80

aDegree of digestion was measured gravimetrically after the organism was inoculated into 100 ml of media containing each substrate as a sole source of carbon. Inoculum was incubated for 5 days at 30 C on reciprocal shaker.
TABLE 3. Effect of alkali treatment on the digestibility of bagasse by the organism
Treatment

N W

60 40

_i 0
0.01

D 04

wO

20

digestiona
NaOH

Degree of

Temp

Time
min

FIG. 2. Effect ofpH on the growth of the organism and the activity of the extracellular cellulase.

No

losic materials. As shown in Table 2, the regenerated cellulose, such as filter paper and paper towels, as well as alkali-treated bagasse was easily digested, whereas cellulose, such as cotton fiber and untreated bagasse, was difficult to digest or was not digested at all. Thus, it is apparent that by chemical or physical treatment, or both, the structural features of cellulose fibers are altered so that it becomes susceptible to the enzymatic

2 2 2 10 10 30 30 50 50

80 80 100 100 25 80 100 100 25

30
15 15 30 90 30 15 90 90

% 44 42 43 43 35 53 59 89 79

a Degree of digestion was measured gravimetrically after 5 days of incubation.

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was not found in detectable amounts in the medium in which the organism was grown. Therefore, it was presumed that the extracellular enzymes of the organism hydrolyzed cellulose into

cellobiose, which was then further metabolized inside the cell. Referring to the multiple components in ceUulolytic systems of Reese (8), only C1 and C. components, whose specific actions
are to form linear chains of anhydroglucose units and cellobiose, respectively, were present in the

extracellular enzymes of the organism. The fglucosidase, whose specific action is the formation of glucose from cellobiose, may be a mural or intracellular component of the organism. .. The essential amino acid content of the cell protein of the organism was determined, and the values obtained were compared with that of Food and Agricultural Organization (FAO) reference protein pattern (6) and with proteins of other plant and animal sources. As shown in Table 4, the essential amino acid pattern of the cell protein compares favorably with that of FAO reference protein. The lysine content, which is deficient in a number of foods, particularly cereal grains, was higher than that of the reference protein. Other essential amino acid contents, such as leucine and valine, were extremely high in comparison with the proteins of FIG 3 Paper chromatography of the hydrolysate of other sources and the FAO reference protein. filter paper. (1) Glucose and cellobiose standard, (2) filter paper digested by crude enzyme, (3) crude enzyme, ACKNOWLEDGMENT (4) supernatant fluid of culture in which the organism This investigation was supported by funds from the was grown in the presence of filter paper, (5) supernatant fluid of culture in which the organism was grown Graduate Research Council of Louisiana State University. in the absence offilter paper.
A;%~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~...

TABLE 4. Essential amino acid content of the cell proteina (grams of amino acid per 100 g of

1.

protein)

2.
Amino acid

protein reference flWoheat poenproteinb


4.2 2.2 4.2 7.0 1.9 1.5 5.5

Beefc

Arginine .......... 9.21


2.30 4.74 11.20 Leucine..... Lysine............ 6.84 Methionine ....... 1.86 Phenylalanine ..... 4.36 2.67 Tyrosine ...... Threonine .......5.5.37 Valine ...... 10.71
a

Histidine .......... Isoleucine .........

4.2 4.8 4.2 2.2 2.8 2.8 2.8 4.2

7.7 3.3 6.0 8.0 10.0 3.2 5.0

3.
4.

5.

2.7 4.1

5.0 5.5

The sample was hydrolyzed with 6 N HCl at 110 C for 22 hr and determined with a Beckman model 116 amino acid analyzer. bNational Academy of Science-National Research Council (7). c M. S. Iyengar, Single Cell Protein Conference, Massachusetts Institute of Technology, Cambridge, 1967.

6.

7.

LITERATURE CITED Cifonelli, J. A., and J. Smith. 1954. Detection of glycosides and other carbohydrates on paper chromatography. Anal. Chem. 26:1132-1134. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. Halliwell, G. 1961. The action of cellulolytic enzymes from Myrothecium verrucaria. Biochem. J. 79:185-192. Kitts, W. D., and L. A. Underkofler. 1954. Hydrolytic products of cellulose and the celluloytic enzymes. Agr. Food Chem. 2:639-645. Lembeck, W. J., and A. R. Colmer. 1967. Effect of herbicides on cellulose decomposition by Sporocytophaga myxococcoides. Appl. Microbiol. 15:300-303. National Academy of Science-National Research Council. 1963. Evaluation of protein quality, p. 74. Publication 1100, National Academy of Science-National Research Council, Washington, D.C. Okamoto, T., and T. Asai. 1952. Studies on the aerobic mesophilic cellulose decomposing bacteria in Japan. VI. On the bacterial cellulase. J. Agr. Chem. Soc. (Tokyo) 26:137-140.

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8. Reese, E. T. 1956. Enzymatic hydrolysis of cellulose. Appl. Microbiol. 4:39-45. 9. Society of American Bacteriologists. 1957. Manual of microbiological methods, p. 315. McGraw-Hill Book Co., Inc., New York. 10. Stahl, E. 1961. Dunnschicht-chromatographic

VI. Mitteilung spurenanalyse von Zuckergemischen Auf Kieselgur G-schichten. J. Chromatog. 5:351-355. 11. Youatt, G. 1960. Some factors influencing the breakdown of cellulose by bacteria. Australian J. Biol. Sci. 13:188-195.

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